Vibrational Fingerprint Analysis of an Azo-based Resonance Raman Scattering Probe for Imaging Proton Distribution in Cellular Lysosomes.

Due to the fundamental mechanism of vibrational state transitions for chemical bonds, the spectra of Raman scattering are narrow-banded and photostable signals capable of probing specific reactions. In the case of protonation/deprotonation reactions, certain chemical bonds are broken and new bonds are formed. Based on the changes of the vibrational modes for the corresponding bonds, fingerprint analysis of multiple Raman bands may allow for the in situ visualization of proton distribution in live cells. However, Raman scattering faces the well-known challenge of low sensitivity. To perform the vibrational fingerprint analysis of Raman scattering by overcoming this challenge, we developed an azo-based resonance Raman pH probe. It was an azobenzene-featured small molecule responsive to protons with the inherent Raman signal ∼104-fold more intense than that of the conventional alkyne-type Raman reporter 5-ethynyl-2'-deoxyuridine. Through the substitution of the electron-donating and -withdrawing entities to the azobenzene group, the effect of resonance Raman scattering and fluorescence quenching was obtained. This effect resulted in a significant Raman enhancement factor of ∼103 compared to the counterpart molecules without the molecular design. Based on the enhanced Raman sensitivity of the azo-based resonance Raman pH probe, the identification of vibrational fingerprint changes at the azo group was achieved during the protonation/deprotonation reactions, and the vibrational fingerprint analysis resolved a pH difference of less than 0.2 unit. The method enabled sensitive hyperspectral cell imaging that clearly visualized the change of proton distribution in autophagic cells.